Mutagenesis method

ABSTRACT

This application provides a method for mutagenesis of a gene, which comprises introducing much more point mutations into one strand of double-stranded genomic DNA of cell or organism individual than into another strand. In accordance with such a method, it is now possible to efficiently and effectively construct various useful mutants of microorganisms, cells or organism individuals. It is also now possible by analyzing the mutating conditions of the gene to clarify the mechanism of drug resistance, to estimate the occurrence of a novel insensible microorganism or to develop a drug therefor, to analyze the mutation of an oncogene and the mechanisms of cancer metastasis and increase in malignancy, to develop a therapeutic method using these mechanisms, etc.

TECHNICAL FIELD

[0001] The invention of this application relates to an effective andhighly efficient method for introducing a random mutation in whichmutation can be efficiently introduced into a cell or an organismindividual and also a risk of extinction of treated cell or individualgroups can be reduced, and this invention also relates to mutants andmutated phenotypic gene obtained by the said method.

BACKGROUND ART

[0002] With regard to an art for a genetic modification of cells ororganism individuals, a method where mutagen such as ultraviolet ray,radioactive ray or mutagenic substance is applied to cells or organismindividuals, a method where exogenous gene is introduced into cells ororganism individuals to modify by means of genetic engineering, etc. areavailable. In the case of induction of mutation in specific gene, therehas been known a method where genetic engineering means such assite-specific mutation induction and in vitro mutation induction byaccumulation of replication mistake in DNA utilizing a PCR amplifyingtechnique.

[0003] Generally, when the site into which gene or mutation to bemodified is clear, genetic engineering means may be sometimes effectivewhile, when the knowledge about the phenotype to be modified or genetherefor is insufficient, there is an effective method utilizing arandom mutation induction where mutation is randomly introduced intogene and, from the resulting mutant, cells or organism individualshaving the desired mutation phenotype is selected. In inducing therandom mutation, a method where mutation is induced by irradiation ofultraviolet ray, X-ray or radioactive ray to cells or organismindividuals, a method where mutation is induced by treating with amutagenic substance such as nitrogen mustard or nitrosoguanidine, andthe like are available.

[0004] In the conventional art for introducing a random mutation, themutation rate induced by ultraviolet ray or mutagen has an importantinfluence on efficiency and effect of the treatment. Thus, when theinduced mutation rate is within an optimum range, mutation in asufficient amount for DNA is accumulated while, when it is less than theoptimum amount, mutation may be sometimes repaired by a repairingmechanism, etc. of DNA whereby mutation cannot be introducedefficiently. Further, when it is more than the optimum amount, thelethal effect to the organism by the introduced mutation becomes strongwhereupon, before the desired mutant is obtained, the group into whichmutation is introduced and is treated therewith dies out resulting in noproduction of the desired mutant.

[0005] The same thing may be said for the optimum amount not only justfor one treatment but also for plural and continued treatments wheremutagenetic treatment and mutant selection are carried out one afteranother in order to obtain more highly useful mutant. Thus, unless theoptimum amount is determined carefully, the efficiency is bad or thegroup subjected to a mutagenesis dies out whereby it is at lastimpossible to obtain a highly useful mutant. In addition, there may be acase where it is necessary to introduce plural mutations into a gene inwhich phenotype of cells or organism individuals to be modified by arandom introduction of mutation is single or a case where it isnecessary to introduce mutation into plural genes and, in such cases,randommutation is to be inserted until preferred mutation is accumulatedin such genes. However, accumulation of many mutations results in a highrisk where lethal mutation is introduced into the gene which isnecessary for living. Thus, the higher the mutation rate, the more therisk of extinction of the treated cells or organism individuals wherebyit cannot be expected to obtain a useful mutant in an efficient manner.

[0006] In recent years, although the object is not to genetically modifythe cells or the organism individuals, a method has been developed foran efficient introduction of a random mutation into gene inserted into aplasmid in the following mutant of Escherichia coli utilizing the factthat the mutation rate of E. coli simultaneously having mutD, muS andmutT (which are mutator genes concerning proofreading mechanism ofmispair of DNA base pair, A/T-G/C transversion and mismatch repair ofDNA, respectively) is 5,000-fold of a wild type strain (MolecularBiotechnology, 7:189-195, 1997). According to such a method, it ispossible to introduce a mutation for each 1,000 base pairs into genes onplasmid by incubation for 24 generations within about one day. However,although such a high mutation rate increases the probability ofmutagenesis of gene into which mutation is to be introduced, that alsoincreases a risk of introduction of mutation into other genes such asthose necessary for living. Therefore, when the length of the DNA regioninto which mutation is to be introduced is 100 base pairs or less orwhen mutation is to be introduced into plural parts, a PCR isrecommended because the above method is not practical due to an increasein numbers of growth generation. It has been also pointed out that,since the mutation rate is high, incubation of the strain for a longperiod causes an affection to the cell per se or genotype thereofwhereby carefulness is required. Accordingly, although the said methodis suitable for introduction of mutation into gene on plasmid introducedinto a host, it is not suitable for a genetic modification of the hostitself.

[0007] As mentioned above, in the conventional method for theintroduction of a random mutation into cells or organism individuals,introduction of many mutations and avoidance of extinction ofmutation-introduced group are in a relation of antinomy whereby it isdifficult to obtain various and useful mutants in an efficient manner.

[0008] The invention of this application has been achieved in view ofthe above circumstances and its object is to provide a method where arandom mutation is introduced into a cell or an organism individual in ahigh mutation rate and, at the same time, risk of extinction of treatedgroup is reduced and useful and various mutants are efficientlyobtained.

DISCLOSURE OF INVENTION

[0009] As an invention for solving the above-mentioned matters, thisapplication provides a method for mutagenesis of a gene, which comprisesintroducing much more point mutations into one strand of double-strandedgenomic DNA of cell or organism individual than into another strand.

[0010] The first preferred embodiment of the method of the presentinvention is that the point mutation is randomly introduced into fourbases constituting the double-stranded genomic DNA.

[0011] The second preferred embodiment of the method of the presentinvention is that the cell or the organism individual is a mutant cellstrain or a mutant organism individual having mutator gene in a mutationrepair mechanism gene group. Incidentally, such a mutant cell strain ora mutant organism individual may inherently have the mutator gene or maybe that into which extrinsic mutator gene is introduced.

[0012] The third preferred embodiment is that, in the above-mentionedmethod, the mutator gene is one or more mutator gene(s) selected from agroup consisting of dnaQ, dnaE, mutL, mutS, mutH, uvrD and dam.

[0013] The fourth preferred embodiment of the above-mentioned is thatthe mutator gene is a gene which causes a defect of mutation repairmechanism under a certain condition.

[0014] The fifth preferred embodiment of the method is that thecondition for the defect of the mutation repair function is a certaintemperature.

[0015] The sixth preferred embodiment of the method is that a step ofintroduction of mutation into genomic DNA under a certain condition anda step of selection of mutant under a selected pressure conditionwithout introduction of mutation are repeated.

[0016] The seventh preferred embodiment of the method is that the stepof introduction of mutation at the second time and thereafter arecarried out under the same selected pressure as that in the step ofmutant selection immediately therebefore.

[0017] As another invention, this application also provides a mutant ofcell or organism individual where mutation is introduced into genomicDNA by any of the above-mentioned methods, and also provides a mutatedgene which is isolated from the said mutant.

BRIEF DESCRIPTION OF DRAWINGS

[0018]FIG. 1 is ampicillin concentration-survival curve of dnaQ49 strainbefore introduction of mutation and a wild type.

[0019]FIG. 2 is ampicillin concentration-survival curve showingampicillin sensitivity of dnaQ49 strain after the first introduction ofmutation, a wild type and an MNNG wild type.

[0020]FIG. 3 is ampicillin concentration-survival curve showingampicillin sensitivity of dnaQ49 strain after the second introduction ofmutation, a wild type and an MNNG wild type.

[0021]FIG. 4 is ampicillin concentration-survival curve showingampicillin sensitivity of dnaQ49 strain after the third introduction ofmutation, a wild type and an MNNG wild type.

[0022]FIG. 5 is ampicillin concentration-survival curve showingampicillin sensitivity of dnaQ49 strain after the fourth introduction ofmutation, a wild type and an MNNG wild type.

[0023]FIG. 6 is ampicillin concentration-survival curve showingampicillin sensitivity of dnaQ49 strain after the fifth introduction ofmutation, a wild type and an MNNG wild type.

[0024]FIG. 7 is ampicillin concentration-survival curve showingampicillin sensitivity of another dnaQ49 strain after the fifthintroduction of mutation.

[0025]FIG. 8 is an agarose electrophoresis of DNA fragments derived fromampC genes from each of a wild type dnaQ⁺strain and a dnaQ49 strain.

BEST MODE FOR CARRYING OUT THE INVENTION

[0026] Natural mutation is fixed according to the following particulars.First, injury is generated by oxygen radicals or intravital metaboliteswhich physically or chemically affect the chromosomal DNA or erroneousbase pair is resulted by an error or the like in DNA duplication. Injuryand duplication error resulted in chromosomal DNA as such are calledpromutagenic injury and, when the promutagenic injury is not repairedduring the next DNA duplication, it is fixed in the chromosomal DNA asmutation. Most of the causes of generation of promutagenic injury innatural mutation are duplication error while the ratio of injury ofchromosomal DNA affected by physical and chemical causes is small (CRC,1(3):140-148, 1992). Duplication error is resulted by formation oferroneous base pair depending upon tautomerism of four kinds of basepairs during the DNA synthesis and its frequency is from 10⁻⁴ to 10⁻⁵(Molecular Biology of the Cell, Garland Publish Inc., New York & London,224-225, 1983) On the other hand, there are various mutation repairfunctions in cells whereby, due to mismatch repair system andproofreading function of DNA synthesizing enzyme, etc., the promutagenicinjury is repaired to an extent of about 10⁻³ each and, finally, thefrequency of fixation of the point mutation by a base substitutionlowers to an extent of 10⁻¹⁰ to 10⁻¹¹ per base (CRC, 1(3):140-148,1992). Besides the above, there is a frameshift mutation by insertionand deletion of bases but its frequency is very little.

[0027] In the conventional random mutagenesis treatment, promutagenicinjury is increased in chromosomal DNA by enhancing physical andchemical affection by energy rays such as radioactive ray andultraviolet ray or by treatment with mutagen so as to increase theprobability of fixation of the said mutation. Each of the type ofmutagen and the promutagenic injury induced thereby has a characteristicfeature and it is said that, for example, in the case of ultravioletray, both pointmutation and deletion mutation take place to the sameextent while, in the case of point mutation, transition mutation in thedirection of G/C-A/T often occurs. In addition, in the case of X-ray,there are a single strand break where only one of double strands of theDNA chains is cleaved and a double strand break where both are cleavedand, as a result, point mutation and deletion mutation occur where adeletion mutation is apt to take place as much as ten-fold as comparedwith a point mutation (Bunshi Hoshasen Seibutsugaku [MolecularRadioactive Biology] by Sohei Kondo, Tokyo University Press, 138-139,1972).

[0028] On the other hand, in relation with the repair of mutation andduplication error, various mutator genes which increase the mutationhave been known and they may be classified into five according to theirfunctions (CRC, 1(3):140-148, 1992). The first one is mutation ofα-subunit of DNA synthesizing enzyme concerning a control system for DNAduplication error. The thing which has been known up to now is dnaE geneand this mutator gene increases the mutation to an extent of 1,000- to100,000-fold. The second one is mutation of ε-subunit of DNAsynthesizing enzyme participating in proofreading function whichinstantly removes the mispair. With regard to this mutator gene, dnaQ(mutD) has been known and it also increases the mutation to an extent of1,000- to 100,000-fold. The third one relates to a mismatch repair forremoval and repair of mispair which was unable to be corrected by aproofreading function and mutL, mutS, mutH, uvrD, dam, mutY, mutM andung have been known where each of them increases the mutation to anextent of 10- to 1,000-fold. The fourth one relates to a defecationfunction of a nucleotide pool used as a DNA material and mutT whichincreases only A/T-G/C transversion has been known. The cell having thismutator gene increases the GC content in DNA together with the growth ofthe cell and induces the mutation to an extent of 1,000- to 10,000-fold.To the last group belong mutA and mutC and they increase all types oftransversion to an extent of about ten-fold although its function hasnot been known so well.

[0029] As such, the source for promutagenic injury which is a cause ofmutation and the mutation which is induced thereby differ depending uponmutagens and it suggests the possibility that the resulting effect isdifferent depending upon the type of mutagen which is used for theinduction of a random mutation. In addition, in order to improve theeffect of a random mutation, it is effective to randomly introduce apoint mutation into all bases rather than to increase the frequency oftransversion of specific base pair and frameshift mutation by insertionor deletion of a base.

[0030] The inventors of this application have carried out a simulationconcerning the mutated numbers and distribution thereof in the casewhere mutation is uniformly introduced into both DNA strands of adouble-stranded DNA (parity) and in the case where mutation is unequallyaccumulated on one of the DNA strands (disparity). The result showsthat, under the condition where one mutation is introduced by onecleavage per genome (parity), a mode of the mutation numbers indistribution of mutation after ten generations is nearly ten in all of12 simulations and the distribution is from 2 to 20 and that, whengeneration alternations are continued, the generation numbers are nearlyin a mode of mutation numbers and the same distribution tends to beshifted. It has been on the other hand found that, under the conditionwhere, although the total mutation rates are same, mutation isintroduced into one strand of the double-stranded DNA in a probabilityof 100-fold or more as compared with another strand thereof (disparity),the mode of the mutation number mode after ten generations is 10 and itsdistribution is from 0 to 24. The above shows that, even when the totalmutation numbers are same, distribution of mutation numbers is differentafter elapse of generations if many mutations are accumulated on one ofthe DNA strands (J. Theoretical Biology, 157:127-133, 1992).

[0031] Those findings by the inventors are important in introducing arandom mutation. When energy ray such as radioactive ray or ultravioletray or mutagen is used as a cause for promutagenic injury, it is likelythat a promutagenic injury is randomly induced to both strands ofdouble-stranded DNA. Distribution of the mutated numbers in that case issame as that in the case of the above-mentioned parity. On the otherhand, many of mutator genes participate in the function of repair ofpromutagenic injury and, therefore, distribution of the fixed mutatednumbers as a result thereof is understood to be as follows. Since mutTrelates to a base-specific repair function of A/T-G/C, mutation is in anincreasing manner to GC base pairs. On the contrary, mutY, mutM and ungare in a reversed base dependency from mutT and, similarly, they are inan increasing manner to AT base pairs. Since dnaE and dnaQ participatein a proofreading function and a control of duplication error in laggingstrand of a double-stranded DNA, they are DNA strand-dependent. AlthoughmutL, mutS, mutH, uvrD and dam participate in a mismatch repair, theyare not specific to DNA strands and, therefore, it is likely that theyreflect the state of promutagenic injury. Similarly, mutA and mutC haveno specificity to bases and DNA strands and participate in all types oftransversion repair.

[0032] Incidentally, the actual effect of mutation is classified intothat which has a lethal effect, that which is neutral having no or verylittle affection to gene functions, and that which gives certain changesto function. Generally, there is a possibility that the mutation showingno lethal effect is conserved. Further, with regard to randomicity ofmutation, mutT, mutY, mutM and ung tend to increase the specific basepair when the function of mutator gene is taken into consideration. Inaddition, some mutagens increase a specific promutagenic injury such asformation of thymidine dimer. Mutagenesis of such a type is presumed tobe insufficient for randomicity and, when that is used for mutagenesis,accumulation of specific mutation is resulted whereby there is apossibility that the effect of genetic improvement of organism isrestricted.

[0033] On the other hand, in the synthesis of DNA, formation oferroneous base pair due to tautomerism of four kinds of bases takesplace randomly for the four bases and, unless the promutagenic injurybased thereupon is repaired, it is likely that a random mutation can beintroduced into chromosomal DNA That which makes the above possible ismutator gene participating in a DNA proofreading function such as dnaEand dnaQ.

[0034] The inventors of this application have further found that, in theplasmid inserted into Escherichia coli having dnaQ which is a mutagenicmutator gene sensitive to temperature, lagging chain has a mutation rateof as high as several-fold to 100-fold as compared with leading chainafter several duplications (Mol. Gen. Genet., 251:657-664, 1996). Theinventors of this application have further found, as shown in Example 4,that more mutations are resulted in lagging chain than in leading chaineven in genomic DNA of dnaQ49 strain. The fact that mutation isunequally accumulated in the double-stranded DNA is presumed to increasethe diversity of mutation unlike in the case of uniform accumulation.

[0035] In order to effectively and efficiently introduce the mutationtogether with reducing the risk of extinction of cells or organismindividuals, it is necessary that the genetic diversity of cells andorganism individuals existing in the treating group is made much moreand, for such a purpose, it is important that point mutations areunequally distributed in the double-stranded DNA together with a randomintroduction of the point mutations into four bases.

[0036] Based upon those findings, the inventors of this application havedeveloped a method for preparing a mutant in efficient and effectivemanner in which more random point mutations are accumulated in one ofDNA strands than in another strand so that the risk of extinction ofmutated cells and organism individuals is reduced together with anincrease in the mutation rate. To be more specific, mutator geneparticipating in a proofreading function is introduced into cells ororganism individuals which are to be genetically modified or improvedand a mutagenesis treatment is carried out under such a condition thatrandom point mutations are accumulated in one of DNA strands in moreamount than in another strand. It is also preferred to use acondition-expressing mutator gene such as temperature sensitivity as amutator gene. When such a mutator gene is used, introduction of mutationand fixation of mutant can be freely set by means of operating thetemperature condition, etc. The mutation rate to be induced ispreferably within a range of 100- to 100,000-fold of natural mutationand the preferred condition is that one of DNA strands is accumulatedwith several fold to 100-fold or even more mutations than another strandis.

[0037] Incidentally, cells and organism individuals having mutator genecan be prepared by a known method (Journal of Bacteriology, 153,1361-1367, 1983). It is also possible that the mutator gene isintroduced by means of genetic engineering.

[0038] It is further possible that a step of introduction of mutationand a step of selection of mutant are carried out separately, the stepof inducing the mutation is carried out under the condition where noselected pressure is applied, a step of fixation and selection of mutantis carried out after the treated individual group is proliferated tocertain numbers and, in the second run and thereafter, the sameoperation is repeated so that the aimed mutant is prepared efficientlyand effectively.

[0039] The invention of this application will now be illustrated by wayof the following examples in more detail and in a specific manneralthough the present invention is not limited by the following examples.

EXAMPLE 1

[0040] HK 1366 (dnaQ49) strain and HK 1370 (dnaQ49) strain havingtemperature-sensitive mutator gene (dnaQ) (received from ProfessorHisaji Maki, Nara Institute of Science and Technology) were incubatedand degree of ampicillin resistance of each strain was measured.

[0041] Incidentally, in the dnaQ49 strain, ε-subunit of DNA polymeraseIII is mutated and there is a defect in a proofreading function of DNApolymerase III. Therefore, DNA duplication error is not able to beproofread whereby mutation is generated in all base substitutions. Sincesuch a mutation is sensitive to temperature, there is an increase in themutation rate from 10⁻⁹ to 10⁻⁴ as a result of a shift of the incubationtemperature from 24° C. to 37° C. On the other hand, a dnaQ+ strain is astrain where the ε-subunit of DNA polymerase becomes normal due to areverse mutation of a dnaQ49 strain. This dnaQ+ strain was used as awild type of the dnaQ49 strain (hereinafter, the dnaQ+ strain may bereferred to as “wild type”)

[0042] First, the wild type and dnaQ49 strain where no mutation wasinduced were planted on an agar medium containing ampicillin of variousconcentrations, incubated at 24° C. for two days to form colonies andampicillin concentration-survival curves for dnaQ49 strain and wild typestrain were prepared. Ampicillin sodium salt (Sigma, A-9518) wasdissolved in pure water and the resulting original solution was added toa culture medium to give a desired diluted concentration. Preparation ofthe concentration-survival curve was carried out by measuring theabsorbance of the medium at 550 nm.

[0043] The result is as shown in FIG. 1 and the dnaQ49 strain was foundto have a slightly stronger ampicillin resistance than the wild type.

[0044] After that, each dnaQ49 strain and wild type was planted in a5-ml L-broth medium in a density of 2,000 cells/ml and incubated for 24hours at 37° C. which was a temperature for mutagenesis. Incidentally,in the case of the wild type, 1-methyl-3-nitro-1-nitrosoguanidine (MNNG;Aldrich 12, 994-1) (one of the known mutagenic substances) was added tothe medium in the concentrations of 0˜60 μg/ml medium.

[0045] The grown cells were recovered after incubation for 24 hours andincubated at 24° C. for 48 hours in a medium of various ampicillinconcentrations and the survival rate of each Escherichia coli wasdetermined by a colony forming method.

[0046]FIG. 2 is ampicillin concentration-survival curve showing theampicillin sensitivity of MNNG wild type, wild type and dnaQ49 strainmutated at 37° C. for 24 hours. The maximum concentration for ampicillinresistance of dnaQ49 strain was 30 μg/ml and was confirmed to besignificantly higher than those of the wild type and the MNNG wild type(3˜6 μg/ml) . Incidentally, the wild type which was incubated in thepresence of MNNG in the concentration of 60 μg/ml did not grow at alland, accordingly, it is believed that MNNG in a high concentrationcauses mutation even to the gene necessary for living whereby anextinction is resulted.

[0047] After that, the second mutagenesis was carried out using thecolonies of the medium containing the maximum concentration ofampicillin grown in the above-mentioned incubation at 24° C. Thus,incubation was carried out at 24° C. in a medium containing 30 μg/ml ofampicillin for dnaQ49 strain, in a medium containing 6 μg/ml ofampicillin for a group treated with 10 μg/ml of MNNG, and in a mediumcontaining 3 μg/ml of ampicillin for other MNNG-treated groups and thegrown cells were washed, adjusted to a density of 2,000 cells/ml andincubated at 37° C. for 24 hours. Then, the cells were washed, plantedin a medium containing various concentrations of ampicillin andincubated at 24° C. for 2-3 days to form colonies whereupon the survivalrate of each Escherichia coli was determined. Incidentally, in theMNNG-treated group, the MNNG in the same concentration as in Example 2was treated.

[0048]FIG. 3 is ampicillin concentration-survival curve showing theampicillin sensitivity of each MNNG treated group, wild type and dnaQ49strain where the second mutagenesis was carried out. The maximumconcentration for ampicillin resistance of dnaQ49 strain was 300 μg/mland was confirmed to be significantly higher than those of the wild typeand the MNNG wild type (6˜30 μg/ml). Incidentally, the MNG-treated groupwhich was not shown in FIG. 3 for the result shows that it died out.

[0049] After that, the same operation was repeated and mutagenesis wascarried out up to the fifth run. The ampicillin concentration (maximumconcentration for ampicillin resistance at the colony formation in thepreceding stage) at the proliferation at 24° C. and the days for thecolony formation at 24° C. are as shown in Table 1. FIGS. 4 to 6 arecurves of ampicillin concentration vs. survival after mutagenesis ofthird to fifth runs.

[0050] It is clear from those results that, by the mutagenesis treatmentof up to the fifth run, dnaQ49 strain which grew even in the presence ofabout 6,000 μg/ml of ampicillin was obtained. When the same mutagenesistreatment was carried out in another dnaQ49 strain, an ampicillinresistant strain up to about 10,000g/ml was obtained by five mutagenesistreatments (FIG. 7). Incidentally, there was no dnaQ49 strain which diedout during such an operation. There was no plasmid in those cellswhereby the resistance was shown to be due to mutation of genome gene.TABLE 1 2nd 5th Strain 1st Run Run 3rd Run 4th Run Run (MNNG Concn) ACID AC ID AC ID AC ID AC dnaQ49 30 8 300 6 1000 5 3000 16 6000 Wildtype(0) 3 8 10 6 10 5 10 16 60 Wildtype (1) 3 8 10 6 10 5 10 16 60 Wildtype(3) 3 8 30 6 100 5 300 16 d.o. Wildtype (6) 3 8 6 6 10 5 10 16 30 Wildtype (10) 6 8 d.o. Wild type (30) 3 8 d.o. Wild type (60) d.o.

[0051] When the minimum growth inhibiting concentration (MIC) of variousantibiotic substances to this ampicillin-resistant dnaQ49 strain wasinvestigated, there was a strong resistance to cefotaxime which is aβ-lactam antibiotic substance as same as ampicillin while no resistancewas acquired for antibiotics having different action mechanisms fromampicillin (Table 2). Incidentally, the resistance concentration ofampicillin-resistant Escherichia coli which has been reported up to nowis 1,500 μg/ml and the said resistance is due to plasmid. Whenmutagenesis was carried out at 37° C. without addition of ampicillin, itwas not possible to obtain an ampicillin-resistant microorganism even byten operations.

[0052] As a result, resistant microorganisms showing a resistance toampicillin of high concentrations were able to be acquired within ashort period as compared with the control using mutagen and theresistant microorganisms which were reported already. On the other hand,in the MNNG-treated group, extinction of the microorganism took place bya high-concentration treatment while, by a low-concentration treatment,resistant microorganism of 300 μg/ml was obtained only. TABLE 2Super-Amp- dnaQ Wild-Type resistant dnaQ Ampicillin 2 1 2048 Cefotaxime0.0313 0.0156 64 Chloramphenicol 1 0.5 0.5 Tetracyctine 0.25 0.25 0.25Rifampicin 8 4 2 Streptomycin 1 1 0.5 Nalidixic acid 1 2 0.5 Ofloxacin0.0156 0.0625 0.0156 Super-Stre.-resisting dnaQ Streptomycin 2048Super-Nali.-resisting dnaQ Nalidixic acid 2048 Super-Oflo.-resistingdnaQ Ofioxacin 1024

EXAMPLE 2

[0053] Mutation was introduced into dnaQ49 strain by the same way as inExample 1 to prepare drug-resistant microorganisms to each of ofloxacin,nalidixic acid and streptomycin. As a result, microorganisms resistantto 500 μg/ml of ofloxacin, to 7,000 μg/ml of nalidixic acid and to26,000 μg/ml of streptomycin were obtained as shown in Tables 3-5. TABLE3 Mutagenesis Concentration of Treatment(s) for Ofloxacin (μg/ml)Incubated Days  1 time 0.001 11  2 times 0.01 6  3 times 0.1 9  4 times1 4  5 times 10 3  6 times 30 3  7 times 50 3  8 times 60 3  9 times 703 10 times 80 3 11 times 90 3 12 times 100 7 13 times 120 3 14 times 1323 15 times 144 3 16 times 150 7 17 times 156 3 18 times 168 2 19 times180 2 20 times 210 2 21 times 240 2 22 times 270 2 23 times 300 2 24times 320 6 25 times 330 4 26 times 340 4 27 times 350 3 28 times 360 429 times 370 3 30 times 380 4 31 times 400 2 32 times 425 3 33 times 4503 34 times 475 3 35 times 500 3

[0054] TABLE 4 Mutagenesis Concentration of Treatment(s) for NalidixicAcid (μg/ml) Incubated Days  1 time 1 1  2 times 10 1  3 times 100 28  4times 200 2  5 times 400 3  6 times 600 2  7 times 1000 2  8 times 11001  9 times 1200 1 10 times 1300 1 11 times 1400 1 12 times 1600 1 13times 1800 2 14 times 2000 1 15 times 2500 1 16 times 3000 1 17 times4000 1 18 times 5000 9 19 times 6000 5 20 times 6200 11 21 times 6400 722 times 6600 5 23 times 6800 5 24 times 7000

[0055] TABLE 5 Mutagenesis Concentration of Treatment(s) forStreptomycin (μ/ml) Incubated Days  1 time 1 3  2 times 10 1  3 times100 28  4 times 1000 2  5 times 3000 2  6 times 4000 2  7 times 6000 2 8 times 8000 2  9 times 9000 3 10 times 10000 4 11 times 12000 4 12times 14000 8 13 times 16000 5 14 times 17000 11 15 times 18000 3 16times 19000 6 17 times 20000 5 18 times 21000 5 19 times 22000 6 20times 23000 4 21 times 24000 8 22 times 25000 10 23 times 26000

[0056] In addition, when mutation of the enzyme relating to acquisitionof resistance of microorganism was analyzed in the ofloxacin-resistantmicroorganism, serine at the position 83 of gyrase A was mutated toleucine in the microorganism having a low resistance (1-30 μg/ml). Inthe microorganism having a resistance degree of 100 μg/ml, serine at theposition 83 of gyrase A was mutated to leucine and, in addition, serineat the position 80 of topoisomerase IV which is another enzyme necessaryfor increasing the resistance was mutated to arginine (Table 6). Fromthat result, it has been shown that, according to the method of thepresent invention, an efficient mutagenesis is possible into pluralgenes. Further, the introduced mutation is the same as that of theresistant microorganism which is clinically observed and the mechanismfor resistance acquisition where the fixed mutation increase togetherwith an increase in resistant property was the same as well (J. Infect.Chemotherapy, 3:128-138, 1997).

[0057] The above result shows that the method of the present inventionis able to simultaneously modify the plural genes relating to theexpression of various biological functions and that such variousmodifications of gene can be utilized for prediction of appearance ofnew mutant such as drug-resistant microorganism and also forclarification of its mechanism, etc. TABLE 6 Degree of Resistance ofMicroorganism (OFLX) GyrA ParC 0.1 μg/ml no no 1.0 μg/ml 83-Ser→Leu no3.0 μg/ml 83-Ser→Leu no 30.0 μg/ml 83-Ser→Leu no 100.0 μg/ml 83-Ser→*Leu83-Ser→Arg

EXAMPLE 3

[0058] Mutation was introduced into dnaQ49 strain by the same way as inExample 1 to prepare an alkali-resistant microorganism. The result isthat, as shown in Table 7, a resistant microorganism up to pH 9.8 wasobtained by mutagenesis for 12 times. TABLE 7 Mutagenesis Treatment(s)for pH Incubated Days  1 time 9.5 2  2 times 9.4 2  3 times 9.4 3  4times 9.4 2  5 times 9.5 3  6 times 9.5 2  7 times 9.5 2  8 times 9.7 39 9 times 9.7 2 10 times 9.7 3 11 times 9.7 16 12 times 9.8

EXAMPLE 4

[0059] It has been known that a codon “att” at the positions 4249-4251of ampC gene of Escherichia coli (GenBank Accession No. J01611, J01583)is apt to be mutated to “ttt”. Now, it was investigated whether themutation of this site was caused by mutation of leading chain or bymutation of lagging chain.

[0060] 1. Methods

[0061] Since it has been known that ampC gene of E. coli is synthesizedas a lagging chain, a-chain plus addition sequence (SEQ ID NO: 1) at thepositions 4260-4212 was used as a probe for measuring the fidelity oflagging chain. In addition, a+ chain plus addition sequence (SEQ ID NO:2) at the positions 4235-4281 was used as a probe for measuring thefidelity of leading chain.

[0062] Further, an oligonucleotide of SEQ ID NO: 3 was used as a linkerDNA for measuring the fidelity of lagging chain and leading chain.

[0063] First, dnaQ49 was incubated at 37° C. for a period of onegeneration (time required for OD₅₅₀ of a microorganism suspensionbecomes two-fold) and then genomic DNA was purified and treated withrestriction enzymes Fnu4HI and MspI to give a double-stranded DNAfragment (positions 4212-4279) including ampC genes.

[0064] The resulting DNA fragment was hybridized with a probe formeasuring the fidelity of lagging chain and a linker DNA. Similarly, theDNA fragment was hybridized with a probe for measuring the fidelity ofleading chain and a linker DNA.

[0065] The DNA fragment and the linker DNA hybridized with each probewere subjected to a ligation using T4 DNA ligase to purify the DNAfollowed by treating with a restriction enzyme TSPE I recognizing the“aatt”.

[0066] After that, the DNA fragment hybridized with the probe formeasuring the fidelity of lagging chain was subjected to a PCRamplification using each of an oligonucleotide having a base sequence (−chain of 4279-4259) of SEQ ID NO: 4 and a linker DNA (SEQ ID NO: 2) as aprimer. In addition, the DNA fragment hybridized with the probe formeasuring the fidelity of leading chain was subjected to a PCRamplification using each of an oligonucleotide having a base sequence ofSEQ ID NO: 5 and a linker DNA as primers.

[0067] Incidentally, as a control, mutation of lagging chain and leadingchain was investigated by the same way for ampC gene of a wild typednaQ+ strain as well.

[0068] 2. Results

[0069] Result of the PCR amplification is as shown in FIG. 8. The saidFIG. 8 is a result of analysis of each PCR product by means of anagarose electrophoresis where lane 1 is the result of electrophoresis ofthe PCR product of a marker and lanes 2 and 3 are the results ofelectrophoresis of the PCR products of a DNA fragment derived from dnaQ+strain ampC gene. As being obvious from the bands of the lanes 2 and 3,no band per 100 bp was observed in the DNA fragment (lane 2) hybridizedwith the probe for measuring the lagging chain and in the DNA fragment(lane 3) hybridized with the probe for measuring the leading chain. Sucha fact means that, in the ampC gene of dnaQ+strain, no mutation tookplace both in lagging chain and leading chain. Thus, the codon “att” wasnot mutated in any of the DNA fragments and, therefore, cleavage by therestriction enzyme TSPE I before the PCR took place whereby no PCRamplification was resulted.

[0070] On the other hand, lanes 4 and 5 are the result where the PCRproducts of DNA fragment derived from dnaQ49 strain ampC genes. As beingobvious from the result shown in the lanes 4 and 5, no band showing thepresence of a PCR product of 100 bp was observed in the DNA fragment(lane 5) hybridized with a probe for measuring the leading chain (i.e.,no mutation took place in the leading chain) while, in the case of theDNA fragment (lane 4) hybridized with a probe for measuring the laggingchain, a band showing the presence of a PCR product of 100 bp wasobserved.

[0071] From the above result, it has been confirmed that, in the case ofEscherichia coli dnaQ49 strain, much more mutations are accumulated inthe lagging chain than in the leading chain even in the level of genomicDNA.

Industrial Applicability

[0072] In accordance with the invention of this application, it is nowpossible to efficiently and effectively construct various useful mutantsof microorganisms, cells or organism individuals. It is also nowpossible by analyzing the mutating conditions of the gene to clarify themechanism of drug resistance, to estimate the occurrence of a novelinsensible microorganism or to develop a drug therefor, to analyze themutation of an oncogene and the mechanisms of cancer metastasis andincrease in malignancy, to develop a therapeutic method using thesemechanisms, etc.

1 5 1 59 DNA Artificial Sequence Description of Artificial SequenceSYNTHESIZED OLIGONUCLEOTIDE 1 aagcggggta attgtgcgat gcacaatatcgttgatttgt tgaggggcac ccccccccc 59 2 57 DNA Artificial SequenceDescription of Artificial Sequence SYNTHESIZED OLIGONUCLEOTIDE 2ttgtgcatcg cacaattacc ccgcttatag agcaacaaaa gatcccgccc ccccccc 57 3 30DNA Artificial Sequence Description of Artificial Sequence SYNTHESIZEDOLIGONUCLEOTIDE 3 gattaggatc cactaatatc gggggggggg 30 4 21 DNAArtificial Sequence Description of Artificial Sequence SYNTHESIZEDOLIGONUCLEOTIDE 4 ggatcttttg ttgctctata a 21 5 21 DNA ArtificialSequence Description of Artificial Sequence SYNTHESIZED OLIGONUCLEOTIDE5 tgcccctcaa caaatcaacg a 21

1. A method for mutagenesis of a gene, which comprises introducing muchmore point mutations into one strand of double-stranded genomic DNA ofcell or organism individual than into another strand.
 2. The methodaccording to claim 1, wherein the point mutations are randomlyintroduced into four kinds of bases.
 3. The method according to claim 1or 2, wherein the cell or the organism individual is mutant cell strainor mutant organism individual having mutator gene in a mutation repairgene group.
 4. The method mutation according to claim 3, wherein themutator gene is one or more mutator genes selected from a groupconsisting of dnaQ, dnaE, mutL, mutS, mutH, uvrD and dam.
 5. The methodaccording to claim 3 or 4, wherein the mutator gene is a gene whichcauses a defect of mutation repair mechanism under a certain condition.6. The method according to claim 5, wherein the condition for the defectof the mutation repair mechanism is a certain temperature.
 7. The methodaccording to claim 5 or 6, wherein a step of introduction of mutationinto genomic DNA under a certain condition and a step of selection ofmutant under a selection load condition without introduction of mutationare repeated.
 8. The method according to claim 7, wherein the step ofintroduction of mutation at the second time and thereafter are carriedout under the same selection load as that in the step of mutantselection immediately therebefore.
 9. A mutant of cell or organismindividual where mutation is introduced into genomic DNA by any of themethods of claims 1 to
 8. 10. A mutated gene which is isolated from themutant of claim 9.